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PubMed1996

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29.

8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca2+ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of "Ca2+ signalling" in single fura-2 loaded HT29 colonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mumol/l intracellular Ca2+ ([Ca2+]i) transient with an IC50 of 20 mumol/l. However, [Ca2+]i transients induced by other phospholipase C coupled agonists ATP (10 mumol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mumol/l). The agonist-induced [Ca2+]i transients remained equally unaffected by 100 mumol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mumol/l for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca2+]i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mumol/l) alone induced a small [Ca2+]i increase (delta[Ca2+]i: 40 +/- 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization (delta pH: 0.1 +/- 0.02, n = 7) occurring simultaneously with the increase in [Ca2+]i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the "tool" TMB-8 as an "intracellular Ca2+ antagonist"; (2) TMB-8 acts a muscarinic receptor antagonist at the M3 receptor; (3) TMB-8 does not influence the release of Ca2+ from intracellular stores when IP3 signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca2+]i transients at higher concentrations.

Adenosine Triphosphate↗
PubMed1998

Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment.

Up-regulation of CD44 variant isoforms has been linked to the progression of epithelial tumors and the metastatic phenotype. Here we report a functional role for CD44 variant isoforms in colorectal cancer metastasis. An antisense mRNA approach was used to down-regulate CD44 variant isoforms containing CD44 variant 6 (v6) in the metastatic colorectal tumor cell line HT29. Cell lines stably expressing antisense CD44 exon 10 (v6) showed reduced expression of alternatively spliced CD44 variant isoforms but no significant change in expression of CD44 core protein, as judged by immunohistochemical analysis using CD44 domain-specific monoclonal antibodies. Expression of antisense exon 10 (v6) had no effect on HT29 tumor cell proliferation in vitro or the ability of the cells to bind immobilized hyaluronan, but it resulted in a reduced capacity to form liver metastases in nude mice following intrasplenic injection. Metastases were not detected in nude mice inoculated with antisense CD44 exon 10 (v6)-expressing cell lines after 4 months, against a background of a 30% metastasis rate in the control HT29 parental and vector alone transfected lines. Furthermore, whereas 82% of mice intrasplenically injected with control HT29 parental and vector alone cell lines developed tumors in incisional wound sites, none of the mice injected with antisense exon 10 expressing HT29 cells developed similar tumors. This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecule generated through alternative mRNA splicing while allowing expression of core domains to remain unaffected. Furthermore, these results provide direct evidence for a functional role of CD44 variant isoforms in the metastasis of human colorectal tumor cells and may suggest a critical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.

Adenocarcinoma↗
PubMed1989

Proliferation of the human colon carcinoma cell line HT29: autocrine growth and deregulated expression of the c-myc oncogene.

Human colon adenocarcinoma cell (line HT29) are able to proliferate in a defined (serum-free) medium containing no added growth factors; in such conditions, their doubling time is 3 to 4 days (on serum-coated dishes) or 2 to 3 days (on an autologous extracellular matrix) compared with 1 day in the presence of fetal calf serum. In the presence of suramin, a polyanion disrupting the binding of growth factors to their receptors, the incorporation of [3H]thymidine in serum-free cultures is reduced (27.0 +/- 2.9% of control after 3 days of culture), suggesting involvement of autocrine growth factors in the autonomous proliferation of the cells. The expression of the proliferation-related oncogene c-myc was examined during various stages of growth and differentiation of the HT29 cells. The cellular contents of c-myc mRNA were similar in all experimental conditions studied: exponential phase; stationary phase; nondifferentiated as well as differentiated cells (by glucose deprivation); and also in serum-free medium containing or not suramin. An approximately 2-fold increase in the level of c-myc mRNA was observed in cells cultured for 3 days in suramin-containing medium and then incubated during 3 h in the absence of suramin (with or without 10% fetal calf serum). Southern blot analysis of the genomic DNA of HT29 cells did not reveal any rearrangement within the region containing the c-myc gene and the flanking sequences (approximately five kilobases upstream and approximately three kilobases downstream). The c-myc locus was weakly amplified (four to six copies per cell). These results indicate that the c-myc gene expression in HT29 cells is deregulated and does not require growth factor stimulation. The deregulation of the c-myc gene may be related to the reduced growth factor requirement of the HT29 cell line.

Blotting, Northern↗
PubMed1990

CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29-D4).

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29-D4) derived from a human colon adenocarcinoma cell line. HT29-D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti-CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29-D4 cells can be induced to differentiate into enterocyte-like cells by removing glucose from the culture medium. Under these conditions, HT29-D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29-D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29-D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.

Antibodies, Monoclonal↗
PubMed1995

Patterns of expression of lineage-specific markers during the in vitro-induced differentiation of HT29 colon carcinoma cells.

The four different cell types present in the mature colon, absorptive enterocytes, mucus-secreting goblet cells, Paneth cells, and enteroendocrine cells, are believed to derive from a common precursor, the stem cell, anchored near the base of the crypt. Stem cell descendants undergo several rounds of cell division, creating a pool of transit cells that are committed to differentiation. The mechanisms by which committed cells are allocated to the different cell lineages of the intestine are poorly understood. We have used the colon carcinoma cell line HT29 and Cl.16E cells, a clonal derivative of HT29 cells, to investigate the regulation and pattern of expression of several markers (MUC2, MUC3, carcinoembryonic antigen, and alkaline phosphatase) that are associated with a more differentiated phenotype and that, in the mature cells, are lineage restricted. HT29 cells can express, upon exposure to the appropriate inducers, distinct intestinal specific markers; they are, therefore, considered multipotent, similar to the stem cells of the crypt. Conversely, Cl.16E cells are lineage restricted and respond to cell contact inhibition by expressing a fully differentiated goblet cell phenotype. We show that, in HT29 cells, different inducers (12-O-tetradecanoylphorbol-13-acetate, forskolin, and sodium butyrate) modulate specific sets of markers. Forskolin induces the expression of both MUC2 and MUC3, whereas 12-O-tetradecanoylphorbol-13-acetate is capable of inducing only MUC2, and sodium butyrate, only MUC3 gene expression. Carcinoembryonic antigen, a marker common to enterocytes and goblet cells; can be induced by all the agents, whereas the alkaline phosphatase gene, the expression of which is characteristic of enterocytes, is responsive solely to sodium butyrate treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1996

Quinone-induced apoptosis in human colon adenocarcinoma cells via DT-diaphorase mediated bioactivation.

DT-diaphorase (DTD) activity has been related to bioactivation and cytotoxicity of antitumor quinones. A pair of human colon adenocarcinoma cell lines, HT29 and BE, were used in this study to examine the role of DTD in antitumor quinone induced apoptosis. HT29 cells have elevated levels of DTD whereas BE cells lack functional DTD due to a point mutation which results in a complete lack of DTD activity. MeDZQ, a quinone that is efficiently bioactivated by DTD, induced apoptosis both in HT29 and BE cells, but with a much higher incidence in HT29, as assessed by morphological criteria and the formation of oligonucleosomal fragments of DNA. Two other quinone compounds which are also substrates for DTD, i.e. streptonigrin and mitomycin C, also preferentially induced apoptosis in HT29 cells, which could be inhibited by dicoumarol. Our data suggest that bioreductive activation of antitumor quinones by DTD results in induction of apoptosis in human colon carcinoma cells.

Adenocarcinoma↗
PubMed2006

Antioxidant levels and inhibition of cancer cell proliferation in vitro by extracts from organically and conventionally cultivated strawberries.

The effects of extracts from five cultivars of strawberries on the proliferation of colon cancer cells HT29 and breast cancer cells MCF-7 were investigated, and possible correlations with the levels of several antioxidants were analyzed. In addition, the effects of organic cultivation compared to conventional cultivation on the content of antioxidants in the strawberries and strawberry extracts on the cancer cell proliferation were investigated. The ratio of ascorbate to dehydroascorbate was significantly higher in the organically cultivated strawberries. The strawberry extracts decreased the proliferation of both HT29 cells and MCF-7 cells in a dose-dependent way. The inhibitory effect for the highest concentration of the extracts was in the range of 41-63% (average 53%) inhibition compared to controls for the HT29 cells and 26-56% (average 43%) for MCF-7 cells. The extracts from organically grown strawberries had a higher antiproliferative activity for both cell types at the highest concentration than the conventionally grown, and this might indicate a higher content of secondary metabolites with anticarcinogenic properties in the organically grown strawberries. For HT29 cells, there was a negative correlation at the highest extract concentration between the content of ascorbate or vitamin C and cancer cell proliferation, whereas for MCF-7 cells, a high ratio of ascorbate to dehydroascorbate correlated with a higher inhibition of cell proliferation at the second highest concentration. The significance of the effect of ascorbate on cancer cell proliferation might lie in a synergistic action with other compounds.

Antioxidants↗
PubMed1988

Photoaffinity labelling of the vasoactive-intestinal-peptide-binding site on intact human colonic adenocarcinoma cell line HT29-D4. Synthesis and use of photosensitive vasoactive-intestinal-peptide derivatives.

N-Hydroxysuccinimidyl 4-azidobenzoate, a u.v.-sensitive heterobifunctional reagent, was used to synthesize photoreactive derivatives of the vasoactive intestinal peptide (VIP). Products of the reaction were purified by reverse-phase h.p.l.c. Three 4-azidobenzoyl-VIP (4-AB-VIP) derivatives were able to compete with monoiodinated 125I-VIP with an apparent KD of 2.5, 6.3 and 12.5 nM compared with 0.6 nM for native VIP. H.p.l.c.-purified mono[125I]iodinated VIP was used to synthesize 4-AB-125I-VIP derivatives. They were used to photoaffinity-label the VIP-binding site of HT29-D4 cells, a clone derived from the human colonic adenocarcinoma cell line HT29. Only one polypeptide, of Mr 70,000 +/- 5000 (mean +/- S.D.) was specifically labelled. The Mr of the component thus characterized was slightly higher than that of the major species (Mr 67,000) labelled after cross-linking experiments using 125I-VIP, conventional homobifunctional reagents and HT29 cells. Nevertheless, the specificity and extent of glycosylation of these two components were identical. These new photosensitive VIP derivatives should be useful tools with which to investigate further VIP-receptor structure and metabolism.

Affinity Labels↗
PubMed1995

The influence of tumour necrosis factor-alpha, interleukin-1 beta and interferon-gamma on the expression and function of the complement regulatory protein CD59 on the human colonic adenocarcinoma cell line HT29.

CD59 is a 18-25 kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up-regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose-dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-alpha and interleukin-1 beta. A dose-dependent increase in the level of CD59 expression was also seen using low concentrations of interferon-gamma (IFN-gamma), while CD59 expression on cells cultured with high IFN-gamma concentrations was comparable to non-stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non-stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape.

Adenocarcinoma↗
PubMed1998

Geldanamycin-induced cytotoxicity in human colon-cancer cell lines: evidence against the involvement of c-Src or DT-diaphorase.

We investigated two of the major proposed modes of action of the benzoquinoid ansamycin geldanamycin using a pair of human colon-carcinoma cell lines, BE and HT29. One potential mechanism of action in colorectal cancer is the inhibition of c-Src kinase activity, since this proto-oncogene is hyperexpressed in human large-bowel tumours. Our results show that despite the 9-fold higher level of c-Src kinase activity found in HT29 cells, there was only a 1.4-fold difference in cytotoxicity as compared with BE cells, the latter being the most sensitive. Moreover, even at concentrations of geldanamycin that resulted in cell kill of 80% or more after a 24-h period of exposure, there was no effect on c-Src kinase activity in HT29 cells, although c-Src protein was depleted at supralethal levels of exposure. We also investigated the metabolism of the quinone moiety of geldanamycin by DT-diaphorase, an enzyme that activates certain quinone antibiotics such as mitomycin C and is hyperexpressed in colorectal cancer cells. Geldanamycin was shown to be a substrate for DT-diaphorase present in HT29 cells. However, the lack of a major differential in cytotoxicity between HT29 and BE cells indicates that this is unlikely to be pharmacologically significant, since the former contains high levels of enzyme activity, whereas BE cells have no significant activity due to a point mutation in the DT-diaphorase (NQO1) gene. Although reduction of geldanamycin was also catalysed by non-DT-diaphorase reductases in HT29 and BE cells, providing the potential for free radical induction, this is unlikely to be significant since studies previously reported by us elsewhere showed that cells exposed to geldanamycin exhibited no evidence of DNA damage. Thus, as far as the mode of action of geldanamycin in human colon-carcinoma cells is concerned, the present results rule out two major possibilities, namely, the involvement of c-Src tyrosine kinase inhibition and DT-diaphorase metabolism.

Antibiotics, Antineoplastic↗
PubMed2004

Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels.

The effects of 10 different extracts of fruits and berries on cell proliferation of colon cancer cells HT29 and breast cancer cells MCF-7 were investigated. The fruits and berries used were rosehips, blueberries, black currant, black chokeberries, apple, sea buckthorn, plum, lingonberries, cherries, and raspberries. The extracts decreased the proliferation of both colon cancer cells HT29 and breast cancer cells MCF-7, and the effect was concentration dependent. The inhibition effect for the highest concentration of the extracts varied 2-3-fold among the species, and it was in the ranges of 46-74% (average = 62%) for the HT29 cells and 24-68% (average = 52%) for the MCF-7 cells. There were great differences in the content of the analyzed antioxidants in the extracts. The level of the vitamin C content varied almost 100-fold, and the content of total carotenoids varied almost 150-fold among the species. Also in the composition and content of flavonols, hydroxycinnamic acids, anthocyanins, and phenolics were found great differences among the 10 species. The inhibition of cancer cell proliferation seen in these experiments correlated with levels of some carotenoids and with vitamin C levels, present at levels that can be found in human tissues. The same inhibition of cell proliferation could not be found by ascorbate standard alone. This correlation might indicate a synergistic effect of vitamin C and other substances. In MCF-7 cells, the anthocyanins may contribute to the inhibition of proliferation.

Antioxidants↗
PubMed2001

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A.

The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching.

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.

Colonic Neoplasms↗
PubMed2003

Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29.

In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFalpha-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFalpha (50 ng ml(-1)) in the absence and presence of nicotine (10(-11)-10(-6) M). HT29 cells contained mRNA for beta1, alpha4, alpha5, and alpha7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 +/- 0.51 ng ml(-1) (n = 5). Nicotine significantly inhibited TNFalpha-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10(-7) M (2.39 +/- 0.78 ng ml(-1), n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are alpha7.

Colitis, Ulcerative↗
PubMed1986

VIP receptor activity during HT29-18 cell differentiation and rat intestinal development.

In undifferentiated clonal HT29-18 cells, VIP produced an important (17-fold) and persistent increase in cyclic AMP generation for 10 min. This activation measured at 37 degrees C in the absence of IBMX was transient for 2 min and was considerably reduced (4-fold increase) in enterocyte-like cells. Retrodifferentiation of HT29-18 cells (after substitution of galactose for glucose) resulted in a partial reversion of VIP receptor activity. Cyclic AMP levels reached a peak value at 1 min but remained elevated over basal values for 10 min. Similarly, maturation of intestinal mucosae in 19-day-old rat fetuses and 5 day-old rats was accompanied by a remarkable reduction of VIP receptor activity. Cyclic AMP phosphodiesterase, adenylate cyclase activities and VIP receptor desensitization might explain this phenomenon. In contrast, the potency of VIP and the pharmacological properties of the VIP receptor are unchanged. We therefore assume that VIP receptor activity is an indicator of intestinal cell differentiation and we suggest that this neuropeptide may be a regulator of the normal and tumoral development of the intestinal mucosae in man and rat.

Adenylyl Cyclases↗
PubMed2003

The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro.

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

Amino Acid Motifs↗
PubMed1989

Lactoferrin-binding sites at the surface of HT29-D4 cells. Comparison with transferrin.

The binding of 125I-lactoferrin to HT29-D4 cells, a clone of HT29 cells, was studied and compared to the binding of 125I-transferrin to the same cells. The binding of the two iron-transport proteins is saturable and reversible suggesting the presence of specific receptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, Kd1 of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non-receptor intermediate fashion. The dissociation constant of transferrin and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non-receptor intermediate fashion. After chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular mass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass. These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition experiments were performed with a high molar excess of both ligand proteins did a small percentage of either of the two ligands crossreact with the receptor for the other, possibly due to a structural similarity of the two glycoproteins.

Adenocarcinoma↗
PubMed2006

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate.

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.

Adenocarcinoma↗